JP5905317B2 - Calibration method and apparatus for fine particle sorting apparatus - Google Patents

Description

  The present technology relates to a calibration method in a microparticle sorting apparatus, the apparatus, and calibration particles. More specifically, the present invention relates to a calibration method and the like in a microparticle sorting apparatus that automatically optimizes the optical position and delay time of a microchip or a flow cell and enables high-precision analysis.

  2. Description of the Related Art A microparticle sorting device (for example, a flow cytometer) that optically detects the characteristics of microparticles such as cells and separates and collects only microparticles having predetermined characteristics is known.

  In a flow cytometer, a sample liquid containing cells is sent to a flow channel formed in a flow cell or a microchip, and the cells flowing through the flow channel are irradiated with a laser to detect fluorescence or scattered light generated from the cells. The optical properties of the cells are measured by detecting with. In the flow cytometer, the population (group) determined to satisfy a predetermined condition as a result of the measurement of optical characteristics is also collected separately from the cells.

  In cell sorting (sorting) in a flow cytometer, a fluid stream (droplet flow) is generated by discharging the sample liquid containing cells and the sheath liquid into droplets from the orifice formed in the flow cell or microchip. Let The sample liquid and the sheath liquid are formed into droplets by applying vibration of a predetermined frequency to the orifice by the vibration element. Droplets containing cells are charged and discharged, and the target cells with desired characteristics and other non-target cells are collected in separate collection containers by electrically controlling the direction of movement of each droplet. To do.

  For example, in Patent Document 1, as a microchip type flow cytometer, “a flow path through which a liquid containing microparticles flows, an orifice for discharging the liquid flowing through the flow path to a space outside the chip, , A vibrating element for ejecting liquid droplets at an orifice, a charging means for imparting electric charges to the ejected liquid droplets, and a microparticle flowing through the flow path Optical detection means for detecting optical characteristics, a counter electrode disposed opposite to the moving liquid droplet in the direction of movement of the liquid droplet discharged to the space outside the chip, and passing between the counter electrodes And a microparticle sorting device including two or more containers for collecting the droplets.

  Patent Document 2 describes controlling the position (hereinafter referred to as “break-off point”) where the sample liquid and the sheath liquid ejected from the orifice are formed into droplets (hereinafter referred to as “break-off point”). 1). Each droplet is charged at this break-off point according to the characteristics of the cells contained in the droplet. The time from when the characteristics of a cell are detected in the flow path formed in the flow cell or microchip until the cell reaches the break-off point and the liquid droplet containing the cell is charged is the “delay time. ".

JP 2010-190680 A Special table 2007-532874 gazette

  In order to accurately measure the optical characteristics of the microparticles, the microparticle sorting device accurately positions the flow position of the microparticles in the flow path formed in the flow cell or microchip and the optical axis of the laser. It is necessary to match. Conventionally, this alignment has been performed manually by a user using calibration particles (calibration beads), which requires proficiency and has a problem in reliability and stability. In particular, in the microchip type microparticle measuring apparatus, it is necessary to adjust the optical position each time the microchip is replaced or analyzed, which is very complicated.

  Further, in order to perform accurate sorting in the microparticle sorting apparatus, it is necessary to set a delay time that can give an appropriate charge to the droplet according to the characteristics of the cells contained in the droplet. Conventionally, a user manually sets a delay time so that good sorting is achieved while receiving a droplet to be sorted on a slide and visually observing it. This operation is also very complicated, requires proficiency, and is insufficient in reliability and stability.

  Therefore, the present technology mainly provides a calibration method in a fine particle sorting apparatus capable of automatically and highly accurately adjusting the position of the microchip or the flow cell with respect to the optical axis of the laser and optimizing the delay time. With a purpose.

In order to solve the above-described problem, the present technology provides a liquid feeding procedure for passing a fluid containing two or more kinds of calibration particles having different sizes and fluorescence intensities through a flow path formed in a microchip or a flow cell, and laser irradiation. A first signal acquisition procedure for detecting light generated from the microchip or the flow cell from a plurality of positions on the microchip or the flow cell, and detection intensity of light generated from all types of the calibration particles, preferably And a procedure for specifying a position where the area average value of the integrated values of the detected intensities becomes larger. A calibration method in a microparticle sorting apparatus is provided.
In this calibration method, within the area where the area average value is larger, the detection intensity of light generated from a particle of a small size among the calibration particles, preferably an integrated value or an average value of the detection intensity , And the integrated value of the detection intensity of light generated from particles of a small size among the calibration particles in the area where the area average value becomes larger Or a procedure for specifying a second optimum position where the coefficient of variation of the average value becomes smaller.
Further, the calibration method includes a procedure for setting a relative position of the microchip or the flow cell with respect to the laser to the first optimum position or the second optimum position. When the first optimum position and the second optimum position are different, the relative position is set to the second optimum position.
In this calibration method, the position of the microchip relative to the optical axis of the laser can be automatically optimized by setting the relative position of the microchip relative to the irradiation detection unit to these positions.
Further, in this calibration method, a predetermined time elapses from a detection time of light generated from the calibration particles flowing through the flow path with respect to a droplet discharged from an orifice formed at one end of the flow path. The second signal acquisition procedure for detecting the light generated from the droplet after laser irradiation, the integrated value of the detection intensity of the light generated from all types of the calibration particles, or the size of the calibration particles And a step of specifying an elapsed time when the integrated value of the detection intensity of light generated from a large type of particle becomes larger. In this case, the elapsed time can be set in the microchip-type microparticle sorting apparatus as an optimum delay time.
In this calibration method, it is preferable to use two kinds of calibration particles, that is, particles having a small size and a low fluorescence intensity and particles having a large size and a high fluorescence intensity. In this case, it is preferable that the population of particles having a large size and high fluorescence intensity in the calibration particles is larger than the population of particles having a small size and low fluorescence intensity.

Further, the present technology includes an irradiation detection unit that detects light generated by irradiating a laser to a microchip or a flow cell in which a fluid containing two or more kinds of calibration particles having different sizes and fluorescence intensities is passed through a flow path. A position adjusting unit that changes the relative position of the microchip or the flow cell with respect to the irradiation detection unit, and detection intensity of light generated from all types of the calibration particles, preferably an area average of integrated values of the detection intensity And a control unit that outputs a movement signal to a position where the value becomes larger to the position adjustment unit.
In this fine particle sorting apparatus, the control unit is configured to detect the detection intensity of light generated from a small-sized particle of the calibration particles in an area where the area average value is larger, preferably the detection. A movement signal to the first optimum position where the integrated value or average value of the intensity becomes larger is output to the position adjustment unit.
Alternatively, in the area where the area average value is larger, the control unit has a smaller integrated value or a variation coefficient of the average value of the detection intensity of light generated from the small-sized particles of the calibration particles. A movement signal to the second optimum position is output to the position adjustment unit.
The control unit outputs a movement signal to the second optimum position to the position adjustment unit when the first optimum position is different from the second optimum position.
This fine particle sorting apparatus is configured to apply a laser to a droplet discharged from an orifice formed at one end of the flow path after a predetermined time has elapsed from the detection time of light generated from the calibration particles by the irradiation detection unit. It is preferable to include a light source that emits light and a detection unit that detects light generated from the droplet. In this case, the control unit is an integrated value of the detection intensity of light generated from all types of the calibration particles, or an integrated value of the detection intensity of light generated from a type of particles having a large size among the calibration particles, The elapsed time when becomes larger is set as the delay time.

Furthermore, the present technology also provides calibration particles for a microparticle sorting apparatus including two types of particles, which are small in size and low in fluorescence intensity, and large in size and high in fluorescence intensity.
The calibration particles preferably have a larger population of particles having a larger size and a higher fluorescence intensity than a population of particles having a smaller size and a lower fluorescence intensity.
In this calibration particle, the particle size of the particle having a small size and low fluorescence intensity is, for example, 2 to 4 μm, and the particle size of the particle having a large size and high fluorescence intensity is, for example, 8 to 12 μm.

In the present technology, “microparticles” widely include living body-related microparticles such as cells, microorganisms, and liposomes, or synthetic particles such as latex particles, gel particles, and industrial particles.
Biologically relevant microparticles include chromosomes, liposomes, mitochondria, organelles (organelles) that constitute various cells. Cells include animal cells (such as blood cells) and plant cells. Microorganisms include bacteria such as Escherichia coli, viruses such as tobacco mosaic virus, and fungi such as yeast. Furthermore, biologically relevant microparticles may include biologically relevant polymers such as nucleic acids, proteins, and complexes thereof. The industrial particles may be, for example, an organic or inorganic polymer material, a metal, or the like. Organic polymer materials include polystyrene, styrene / divinylbenzene, polymethyl methacrylate, and the like. Inorganic polymer materials include glass, silica, magnetic materials, and the like. Metals include gold colloid, aluminum and the like. The shape of these fine particles is generally spherical, but may be non-spherical, and the size and mass are not particularly limited.

  According to the present technology, a calibration method in a fine particle sorting apparatus capable of automatically adjusting the position of the microchip or the flow cell with respect to the optical axis of the laser and optimizing the delay time with high accuracy is provided.

It is a figure for demonstrating the structure of the microparticle sorting apparatus 1 (flow cytometer 1) based on this technique comprised as a microchip type | mold flow cytometer. 2 is a diagram for explaining a configuration of a flow cytometer 1. FIG. 2 is a diagram for explaining an example of a configuration of a microchip 2 that can be mounted on a flow cytometer 1. FIG. 4 is a diagram for explaining a configuration of an orifice 21 of the microchip 2. FIG. 4 is a flowchart for explaining control steps for optimizing an optical position in the flow cytometer 1. Is a diagram illustrating control in the signal acquiring step S _{2 ~} area average maximum position determination step S _3. In the signal acquisition step S ₂ and S _5, which is a histogram of the detection signals obtained within a predetermined time at each detection position D. Is a diagram illustrating control in the area average maximum position moving step S _{4 ~} integrated value up location determination step S _6. It is a diagram illustrating control in the coefficient of variation determination step S _7. 4 is a flowchart for explaining control steps for optimizing delay time in the flow cytometer 1;

Hereinafter, preferred embodiments for carrying out the present technology will be described with reference to the drawings. In addition, embodiment described below shows an example of typical embodiment of this technique, and, thereby, the scope of this technique is not interpreted narrowly. The description will be made in the following order.

1. Fine particle fractionation measuring device (1) Irradiation detection part (2) Position adjustment part (3) Vibration element (4) Charging part (5) Droplet detection part (6) Deflection plate (7) Collection container (8) Control part (9) Microchip Calibration method in fine particle fractionation measuring apparatus according to the present technology (A) Optimal position optimization control (1) Calibration bead feeding step S ₁
(2) Signal acquisition step S ₂
(3) Area average value maximum position determination step S ₃
(4) Area average value maximum position moving step S ₄
(5) Signal acquisition step S ₅
(6) Integrated value maximum position determination step S ₆
(7) variation coefficient determining step _{S 7}
(8) Position optimization step S ₈
(B) Delay time optimization control (1) Calibration bead feeding step S ₁₁
(2) signal acquisition step _{S 12}
(3) Optimal elapsed time search step S ₁₃
(4) the delay time setting step _{S 14}

1. 1 and 2 are schematic diagrams for explaining the configuration of a microparticle sorting apparatus 1 (hereinafter also referred to as “flow cytometer 1”) according to the present technology configured as a microchip type flow cytometer. FIG. 3 and 4 show an example of the microchip 2 that can be mounted on the flow cytometer 1. FIG. FIG. 3A is a schematic top view, and FIG. 3B is a schematic cross-sectional view corresponding to a cross section along line PP in FIG. FIG. 4 is a diagram schematically illustrating the configuration of the orifice 21 of the microchip 2, in which (A) is a top view, (B) is a cross-sectional view, and (C) is a front view. FIG. 4B corresponds to a PP cross section in FIG.

(1) Irradiation Detection Unit The flow cytometer 1 includes irradiation detection including a light source 61 that irradiates the microchip 2 with the laser L ₁ and a detector 62 that detects detection target light generated by the irradiation of the laser L _1. A part. The irradiation direction of the laser L ₁ with respect to the microchip 2 (the optical axis of the laser L ₁₎ shown in Figure 1 in the Z-axis positive direction. The light source 61 may be an LD, an LED, or the like.

The laser L ₁ is applied to the cells flowing through the sample channel 22 of the microchip 2. The detector 62 detects the scattered light of the laser L ₁ by the cells and the fluorescence generated when the cells or fluorescent dyes labeled on the cells are excited by the laser L ₁ . In FIG. 1, the fluorescence generated from the cells flowing through the sample flow path 22 is indicated by reference numeral F ₁ .

Irradiation detection unit includes a laser L ₁ emitted from the light source 61 is guided to the cell condenser lens and a dichroic mirror for condensing the illumination system comprising a band-pass filter or the like. The irradiation detection unit is configured by a detection system that collects light to be detected generated from the cells by irradiation of the laser L ₁ and guides the light to the detector 62. The detection system includes, for example, a PMT (photo multiplier tube), an area imaging device such as a CCD or a CMOS device, and the like.

Detected light detected by the detection system of the illumination detection unit is a light generated from the cells by irradiation of the laser L _1, for example, forward scattered light or side scattered light, Rayleigh scattering and Mie scattering or the like of the scattered light Or fluorescence. Fluorescence may be generated from cells or fluorescent dyes labeled on the cells. These detection target lights are converted into electrical signals, which are used for determining the optical characteristics of the cells and automatically adjusting the optical position described later.

(2) Position Adjustment Unit The flow cytometer 1 includes a position adjustment unit 9 that changes the relative position of the microchip 2 with respect to the irradiation detection unit. Position adjusting unit 9, the position of the position and / or the irradiation detecting section of the microchip 2 is moved on the vertical plane (XY plane) with respect to the optical axis of the laser L _1. Accordingly, the position adjusting unit 9 adjusts the position of the microchip 2 with respect to the optical axis of the laser L _1, optimized to the laser L ₁ is irradiated to the flowing position of the cells of the sample flow path 22 .

The position adjustment unit 9 only needs to be capable of moving at least one of the position of the microchip 2 or the position of the irradiation detection unit including the light source 61 and the detector 62 in the X-axis direction and the Y-axis direction. The position adjusting unit 9 is configured by, for example, a stepping motor. The position adjustment unit 9, the relative position of the microchip 2 for irradiation detecting section may be those also moved in the Z axis direction (focus direction of the laser L _1).

(3) Vibrating element The flow cytometer 1 applies vibration to the orifice 21 formed in the microchip 2 to form a droplet of the laminar flow of the sample liquid containing cells and the sheath liquid discharged from the orifice 21. Vibration element 3 to be discharged. The vibration element 3 can be, for example, a piezo element. The discharged droplets are ejected in the positive direction of the arrow Y-axis in the figure as a fluid stream S. In the flow cytometer 1, the microchip 2 is mounted so as to be replaceable.

  In the flow cytometer 1, the vibration element 3 may be configured integrally with the microchip 2, and is disposed on the apparatus side so as to be in contact with the mounted microchip 2. Also good.

(4) Charging Part The droplet discharged from the orifice 21 is given a positive or negative charge by the charging part 41. Some droplets may be uncharged and uncharged. The charging of the droplet is performed by an electrode 42 that is electrically connected to the charging unit 41 and inserted into the sample inlet 23 provided in the microchip 2. The electrode 42 may be inserted into any part of the microchip 2 so as to be in electrical contact with the sample liquid or sheath liquid fed through the flow path.

  The charge is applied to the droplets at a time (T0 + ΔT) when a predetermined delay time (ΔT) elapses from the time (T0) when the cell is detected by the irradiation detection unit and the cell reaches the break-off point. Is called.

(5) drop detection unit also flow cytometer 1 includes a light source 71 for irradiating a laser beam L ₂ in the droplets discharged from the orifice 21, the detection for detecting a detection target light generated by the irradiation of the laser L ₂ And a droplet detection unit including the container 72. The irradiation direction of the laser L ₂ (the optical axis of the laser L ₁₎ for the fluid stream S shown in Figure 2 in the Z-axis positive direction. The light source 71 may be an LD, an LED, or the like. The detector 72 may be a PMT (photo multiplier tube), an area imaging device such as a CCD or a CMOS device, or the like.

The laser L ₂ is irradiated to the liquid droplets in the fluid stream S. The detector 72 detects the scattered light of the laser L ₂ by the cells contained in the droplets and the fluorescence generated when the fluorescent dye labeled on the cells or cells is excited by the laser L ₂ . In Figure 2, shows the fluorescence generated from the droplets by reference character F _2.

Light source 71, from the time (T0) the cells are detected by the irradiation detector, a predetermined time to simulate the delay time ([Delta] T) time after the elapse of (Δt) (T0 + Δt) , the laser beam L ₂ in droplet Irradiate. The detected light to be detected is converted into an electric signal by the detector 72 and used for optimizing a delay time described later.

(6) Deflection Plate Further, the flow cytometer 1 includes a pair of deflection plates 51 and 52 arranged to face each other with the fluid stream S interposed therebetween. The deflecting plates 51 and 52 change the traveling direction of each droplet in the fluid stream S by an electric force acting between the electric charges applied to the droplets. The deflection plates 51 and 52 may be commonly used electrodes. In FIG. 1, the opposing direction of the polarizing plates 51 and 52 is indicated by the X-axis direction.

(7) Recovery Container The fluid stream that has passed between the deflecting plates 51 and 52 is received by any of the recovery container 81, the recovery container 82, or the recovery container 83. For example, when the deflection plate 51 is positively charged and the deflection plate 52 is negatively charged, the droplet charged negatively by the charging unit 41 is collected in the collection container 82 and the positively charged droplet is collected in the collection container 83. The In addition, the liquid droplets that are not charged by the charging unit 41 fly straight without receiving the electric action force from the deflecting plates 51 and 52 and are collected in the collection container 81. In the flow cytometer 1, by controlling the traveling direction of the droplets according to the characteristics of the cells contained in each droplet, the target cells having the desired characteristics and the other non-target cells are separately collected. Can be recovered.

  The collection containers 81, 82, 83 may be general-purpose plastic tubes or glass tubes for experiments. These collection containers are preferably disposed on the flow cytometer 1 in a replaceable manner. Moreover, you may connect the drainage path of the collect | recovered droplet to the thing which receives a non-target cell among collection containers. In the flow cytometer 1, the number of collection containers arranged is not particularly limited. When more than three collection containers are arranged, each droplet is guided to one of the collection containers depending on the presence / absence of the electric acting force between the deflecting plates 51 and 52 and the size thereof. To be.

(8) Control unit, etc. In addition to the above-described configuration, the flow cytometer 1 includes a data analysis unit for determining optical characteristics of cells, a tank unit for storing a sample solution and a sheath solution, and the like, which are included in a normal flow cytometer. The control part 10 etc. for controlling each structure of these are provided.

  The control unit 10 can be configured by a general-purpose computer including a CPU, a memory, a hard disk, and the like. The hard disk stores an OS and a program for executing a control step described below.

(9) Microchip The microchip 2 is formed by bonding the substrate layers 2a and 2b on which the sample channel 22 is formed. Formation of the sample flow path 22 on the substrate layers 2a and 2b can be performed by injection molding of a thermoplastic resin using a mold. As the thermoplastic resin, known plastics can be employed as materials for conventional microchips such as polycarbonate, polymethyl methacrylate resin (PMMA), cyclic polyolefin, polyethylene, polystyrene, polypropylene, and polydimethylsiloxane (PDMS).

  The sample liquid is introduced into the sample inlet 23, merged with the sheath liquid introduced into the sheath inlet 24, and fed through the sample flow path 22. After the sheath liquid introduced from the sheath inlet 24 is divided and fed in two directions, the sample liquid is sandwiched from the two directions at the junction with the sample liquid introduced from the sample inlet 23. Join. As a result, a three-dimensional laminar flow in which the sample liquid laminar flow is located at the center of the sheath liquid laminar flow is formed at the junction.

  Reference numeral 25 denotes a suction flow path for eliminating clogging or bubbles by applying a negative pressure in the sample flow path 22 to temporarily reverse the flow when clogging or bubbles are generated in the sample flow path 22. . A suction outlet 251 connected to a negative pressure source such as a vacuum pump is formed at one end of the suction flow path 25, and the other end is connected to the sample flow path 22 at the communication port 252.

  In the three-dimensional laminar flow, the narrowing portions 261 (see FIG. 3) and 262 (see FIG. 4) formed so that the area of the vertical cross section with respect to the liquid feeding direction is gradually or gradually reduced from the upstream to the downstream of the liquid feeding direction. The laminar flow width is narrowed down. Thereafter, the three-dimensional laminar flow is discharged from an orifice 21 provided at one end of the flow path.

Between the narrowing part 261 and the narrowing part 262 of the sample channel 22, cell characteristic detection is performed. By irradiation detection unit, laser L ₁ to the cell in which flows sent by arranging a medium sample channel 22 in a row in the center of the three-dimensional laminar flow is irradiated, fluorescent F ₁ and the scattered light generated from the cell is detected (See FIG. 3).

  A connecting portion of the sample channel 22 to the orifice 21 is a straight portion 27 formed in a straight line. The straight portion 27 functions to eject the fluid stream S straight from the orifice 21 in the positive direction of the Y axis.

  The three-dimensional laminar flow discharged from the orifice 21 is formed into droplets by the vibration applied to the orifice 21 by the vibration element 3 and ejected as a fluid stream S (see FIG. 1). The orifice 21 opens in the direction of the end face of the substrate layers 2a and 2b, and a notch 211 is provided between the opening position and the end face of the substrate layer. The notch 211 is formed by notching the substrate layers 2 a and 2 b between the opening position of the orifice 21 and the substrate end surface so that the diameter L of the notch 211 is larger than the opening diameter l of the orifice 21. (See FIG. 4C). The diameter L of the notch 211 is desirably formed to be twice or more larger than the opening diameter l of the orifice 21 so as not to hinder the movement of the droplets discharged from the orifice 21.

2. FIG. 5 is a flowchart for explaining the control steps for optimizing the optical position of the microchip 2 in the flow cytometer 1. is there. The control steps are “Calibration beads feeding step S ₁ ”, “Signal acquisition step S ₂ ”, “Area average value maximum position determination step S ₃ ”, “Area average value maximum position movement step S ₄ ”, “Signal acquisition”. This includes the steps of “step S ₅ ”, “integrated value maximum position determination step S ₆ ”, “variation coefficient determination step S ₇ ”, and “position optimization step S ₈ ”. Hereinafter, each procedure will be described.

(1) Calibration beads feeding step S ₁
When the analysis start signal is input by the user, the control unit 10 outputs a movement signal to the position adjustment unit 9, and the position adjustment unit 9 sets the relative position of the microchip 2 with respect to the irradiation detection unit to a preset initial position ( (See FIG. 6, origin O). When the relative position is at the origin O, the laser L ₁ emitted from the irradiation detection unit is emitted to the origin O on the microchip 2. The relative position can be changed by moving at least one of the position of the microchip 2 or the position of the irradiation detection unit including the light source 61 and the detector 62 in the X-axis direction and the Y-axis direction. A case where the position of the microchip 2 is moved will be described as an example.

  Next, the control unit 10 drives the pump of the tank unit that stores the sample liquid and the sheath liquid, and starts feeding the sample liquid and the sheath liquid to the sample inlet 23 and the sheath inlet 24 of the microchip 2. The sample liquid includes two or more kinds of calibration particles (beads) having different sizes and fluorescence intensities. Further, the control unit 10 starts applying vibration to the orifice 21 by the vibration element 3. As a result, the three-dimensional laminar flow of the sample liquid and the sheath liquid ejected from the orifice 21 is discharged as droplets, and a fluid stream S is generated.

  As the calibration beads, a resin composition containing a fluorescent dye formed into a particle shape, or a resin dye fixed on the surface of a resin formed into a particle shape can be used. A general-purpose dye such as PB, FITC, PE, PE-TR, or APCCH may be used as the fluorescent dye. In general, the larger the size of the beads, the higher the fluorescence intensity, but the variation in size and fluorescence intensity increases. Conversely, the smaller the size of the beads, the lower the fluorescence intensity, but the smaller the size and fluorescence intensity variations.

  The calibration beads preferably include two types of beads A, which are small in size and low in fluorescence intensity, and beads B which are large in size and high in fluorescence intensity. Also, the population of beads B is preferably larger than the population of beads A.

As the beads A, for example, the particle diameter is 2 to 4 μm, the concentration in the fluid (sample liquid) is about 5.0 × 10 ⁵ / ml, and the variation (coefficient of variation) in fluorescence intensity is 1.5% or less. Things can be used. Further, as the beads B, for example, those having a particle diameter of 8 to 12 μm, a concentration in the fluid of about 5.0 × 10 ⁶ / ml, and a variation (coefficient of variation) in fluorescence intensity of 2.5% or less. Can be used.

  The calibration beads may include beads having an intermediate size and fluorescence intensity between the beads A and B. Hereinafter, a case where calibration beads in which two kinds of beads A and B are mixed is used will be described as an example.

Sample solution and after liquid feed initiation of the sheath liquid, the control unit 10 outputs a movement signal to the position adjusting unit 9, the position adjusting unit 9 moves the position of the microchip 2 in the reference point D ₀ from the origin O (FIG. (See arrow 6). When the relative position of the microchip 2 with respect to the irradiation detection unit is at the reference point D ₀ , the laser L ₁ emitted from the irradiation detection unit is irradiated to the reference point D ₀ on the microchip 2.

Reference point D _0, the position should cell characteristics detection is performed in the microchip 2 (i.e., the optimum position determined by the steps described below) is pre-set in the vicinity of. More specifically, the reference point D ₀ is set in the vicinity between the narrowing part 261 and the narrowing part 262 of the sample channel 22 (see FIGS. 3 and 4).

(2) Signal acquisition step S ₂ (first signal acquisition step)
In this step S _2, the irradiation detection unit, fluorescence or scattered light generated from a plurality of locations on the microchip 2 including the reference point D ₀ (hereinafter, simply referred to as "fluorescence") is detected is performed. In this step S _2, showing the position on the microchip 2 fluorescence detection is performed by the FIG. 6, reference numeral D. The figure shows an example in which 24 detection positions D including the reference point D ₀ are set, and fluorescence from detection positions D arranged in 8 rows in the X-axis direction and 3 rows in the Y-axis direction is detected. It was.

The region where the detection position D is set includes the sample flow path 22, and as long as this is the case, the number and arrangement of the detection positions D are not particularly limited and are arbitrarily set. The detection positions D are preferably arranged in a lattice pattern in the X-axis direction and the Y-axis direction as shown in the figure. In this case, the arrangement intervals W and H of the detection position D in the X-axis direction and the Y-axis direction are the flow path width (flow path diameter) of the sample flow path 22 and the number M of the detection positions D in the X-axis direction and Y-axis direction. ₁ and N ₁ can be set as appropriate. When the channel width of the sample channel 22 is 70 to 100 μm, the arrangement number M ₁ is 8, and N ₁ is 3, the arrangement intervals W and H are set to 25 and 75 μm, respectively, for example.

The detection of fluorescence is performed for one detection position D for a certain period of time. Fluorescence detected in a certain time is integrated and converted into an electrical signal and output to the control unit 10. Fluorescence detection may be performed by a laser L ₁ are sequentially irradiated by scanning the X-axis and Y-axis directions on the detection position D, and detecting the fluorescence emitted. Alternatively, it may be detected collectively by the fluorescence from the detection position D area imaging element by the irradiation of the laser L _1.

(3) Area average value maximum position determination step S ₃
In this step S _3, the control unit 10 calculates the area average value of the integrated value of the detected intensities for each detection position D, the detection position D to the area average value becomes larger, the detection position D preferably to a maximum value Judge automatically.

“Area average value” means an average of integrated values of detection intensities obtained at one detection position D and a plurality of detection positions D within a predetermined distance range therefrom. In FIG. 6, the area average value is obtained at one detection position D ₁ and detection positions D _{2 to} D ₉ within a distance range of 2 W in the X-axis direction and 2 H in the Y-axis direction from the detection position D _1. The case where it was set as the average of the integrated value of the obtained detection intensity was shown.

  It is assumed that how much distance range from one detection position D is set as the area average value can be appropriately determined according to the channel width (channel diameter) of the sample channel 22 and the arrangement intervals W and H.

The control unit 10 compares the area average values calculated for the respective detection positions D, and determines a detection position D where the area average value becomes larger, preferably a detection position D where the maximum value is obtained. Here, the area average value at the detection position D ₁ will be described assuming that the maximum value.

  The area average value is calculated based on the fluorescence generated from the calibration beads among the fluorescence detected for each detection position D within a predetermined time. FIG. 7 shows a histogram of detection signals within a predetermined time at each detection position D. The bead B has a higher fluorescence intensity than the bead A and has a large variation in fluorescence intensity. The area average value is calculated as the sum of the detection intensities of the fluorescence generated from the beads A and B.

Since fluorescence is strongly generated in the sample flow path 22 through which the calibration beads flow, the region R ₁ formed by connecting the detection positions D _{1 to} D ₉ where the area average value is the maximum value is the area where the sample flow path 22 is formed. It can be regarded as a region to be located. Instead of the “area average value”, the detection intensity itself (peak intensity) obtained at each detection position D, or the average value or integrated value of the detection intensity may be used. The detection position D where the peak intensity, the average value, or the integrated value becomes the maximum value can be regarded as a point close to the sample flow path 22.

(4) Area average value maximum position moving step S ₄
If the area average value detection position D ₁ which is a maximum value is specified, the control unit 10 outputs a movement signal to the position adjusting unit 9, the position adjusting section 9 from the location reference point D ₀ of the microchip 2 It is moved to the detection position _{D 1} (see FIG. 8 arrow).

(5) Signal acquisition step S ₅ (first signal acquisition step)
In this step S _5, the irradiation detecting section, area average value detection of fluorescence emitted from a plurality of locations in the region R ₁ to be the maximum value is performed. The detection position D of fluorescence in this step S ₅ shown in enlarged view of FIG. In the figure, fluorescence is detected from (M ₂ × N ₂ ) detection positions D in which M ₂ rows are arranged in the X-axis direction and N ₂ rows are arranged in the Y-axis direction including the detection position D ₁ where the area average value is the maximum value. The case of doing is shown as an example.

The arrangement intervals w and h of the detection position D in the X-axis direction and the Y-axis direction depend on the flow path width (flow path diameter) of the sample flow path 22 and the number of arrangements M ₂ and N ₂ in the X-axis direction and the Y-axis direction. It can be set appropriately. The number of arrays M ₂ and N ₂ is, for example, 11 and 7, respectively. The column spacings w and h are set to, for example, 5 and 25 μm, respectively. Also in the step S _5, the number and arrangement pattern of the detection position D is not intended to be particularly limited. When the peak intensity, average value, or integrated value obtained at each detection position D is used instead of the “area average value”, an appropriate number of values are included in the region including the detection position D where these values are maximum values. A detection position D is set.

The detection of fluorescence is performed for one detection position D for a certain period of time. The fluorescence detected for a certain time is converted into an electric signal and output to the control unit 10. Detection of fluorescence, the laser L ₁ is scanned in the X-axis and Y-axis directions sequentially scanned on the detection position D, and detecting the fluorescence emitted. Alternatively, the area image sensor, the fluorescence from each of the detection position D by the irradiation of the laser L ₁ may be collectively detected.

(6) Integrated value maximum position determination step S ₆
In this step S _6, the control unit 10, for each detection position D, and calculates the either or both of the accumulation value or average value of the detected intensity, and their coefficient of variation (CV value). Hereinafter, a process using the integrated value of the detected intensity and the CV value will be described as an example.

The control unit 10 compares the integrated values of the detected intensities calculated for the respective detection positions D, and determines the detection position D where the integrated value becomes larger, preferably the detection position D (first optimal position) where the maximum value is obtained. decide. Here, the integrated value at the detection position D ₁₁ will be described assuming that the maximum value (see FIG. 8).

  Calculation of the integrated value of the detection intensity and the coefficient of variation is performed based on the fluorescence generated from the beads A among the fluorescence detected within a predetermined time for each detection position D (see the histogram in FIG. 7). The fluorescence generated from the bead A and the fluorescence generated from the bead B may be distinguished by the difference in the fluorescence wavelength, and only the fluorescence that matches the fluorescence wavelength of the bead A may be detected. Alternatively, the fluorescence generated from the beads A and the fluorescence generated from the beads B can be detected by gating the beads according to the difference in size and detecting only the fluorescence generated from the beads within the selected size range (gate). Can also be distinguished.

(7) variation coefficient determining step _{S 7}
Next, the control unit 10, the integrated value is compared with CV values between the detection position _D 12 _{to D 19} adjacent the detection position _{D 11} with a maximum value smaller than the detection position _{D 11} CV The presence / absence of a detection position D (second optimum position) that gives a value is automatically determined (see FIG. 9).

(8) Position optimization step S ₈
In step S ₇ , a detection position D that gives a smaller CV value than the detection position D ₁₁ where the integrated value is the maximum value is found in any of the detection positions D _{12 to} D ₁₉ adjacent to the detection position D _11. when it becomes, the control unit 10 moves to the detection position D ₁₁ the position of the microchip 2 from the detection position D _1. At this time, the integrated value is consistent with the detection position (first optimum position) and the detection position that the CV value is the minimum value (the second optimum position) Any detection position D ₁₁ with a maximum value.

In step S ₇ , if a detection position D that gives a smaller CV value than any of the detection positions D ₁₁ is found in any of the detection positions D _{12 to} D ₁₉ , the control unit 10 determines the position of the microchip 2. the move from the detection position _{D 1} to the detection position D (for example, the detection position _{D 18).} At this time, the detection position where the integrated value becomes the maximum value (first optimal position) and the detection position where the CV value becomes the minimum value (second optimal position) do not match.

Detecting position D ₁₁ of the integrated value becomes the maximum value, the fluorescence is most strongly generated position, can be regarded as a flowing position of the calibration beads specimen path 22. That is, when the relative position of the microchip 2 with respect to the irradiation detection unit is at the detection position D ₁₁ , the laser L ₁ emitted from the irradiation detection unit is irradiated to the calibration bead passing position of the sample flow path 22. Become.

Exceptionally, the detection position D ₁₁ the integrated value is the maximum value, in less than not the flowing position of the calibration beads of the sample flow path 22, there is a case where the integrated value of the fluorescence intensity is the maximum value . For example, the detection position D ₁₁ is, if it matches the channel walls of the microchannel 22 may unusually high fluorescence intensity and the like reflected or scattered fluorescent from being detected sporadically. In this case, the fluorescence intensity detected at the position varies, and the CV value of the integrated value of the fluorescence intensity increases.

When the detection position D ₁₁ coincides with the flow path wall of the micro flow path 22, the detection position D ₁₈ that gives a smaller CV value among the detection positions adjacent to the detection position D ₁₁ is the calibration of the sample flow path 22. It can be regarded as the flow position of beads. That is, when the relative position of the microchip 2 with respect to the irradiation detection unit is at the detection position D ₁₈ , the laser L ₁ emitted from the irradiation detection unit is irradiated to the calibration bead passing position of the sample flow path 22. Become.

As described above, in the flow cytometer 1, the laser is positioned at a position where the integrated value or average value of the detection intensity of fluorescence generated from the microchip 2 by the irradiation of the laser L ₁ becomes larger, or at a position where the CV value becomes smaller. setting the relative position of the microchip 2 for L _1. Carried out by this, the flow cytometer 1, the flowing position of the cells of the sample flow path 22 of the microchip 2, the optical axis of the laser L _1, a highly accurately positioned automatically, a simple high-precision measurement It is possible.

Further, in the flow cytometer 1, rough adjustment (steps S ₂ and S ₃ ) for specifying a position where the area average value of the integrated value of the fluorescence detection intensity becomes larger, and the integrated value in a region where the area average value becomes larger. or a position where the average value becomes larger, or by two-step procedure with the fine adjustment (step S ₅ to S ₇₎ the CV value to identify a more reduced position, and optimizes the optical position of the microchip 2. As a result, the optical position of the microchip 2 can be optimized quickly with a small processing load.

Further, in the flow cytometer 1, coarse adjustment (steps S ₂ and S ₃ ) is performed based on the detection intensity of light generated from all types of calibration beads (here, beads A and beads B), and fine adjustment ( Steps S _{5 to} S ₇ ) are performed based on the detection intensity of light generated only from a small-sized bead (bead A). Thus, the coarse adjustment can be controlled by using a strong detection signal, the fine adjustment is carried out through flow position of the cells of the sample flow path 22 and the laser L ₁ aligned with the optical axis with high precision be able to. Here, the microchip type flow cytometer in which the flow path is formed in the microchip has been described as an example, but the same effect can be obtained also in the flow cell type flow cytometer in which the flow path is formed in the flow cell.

(B) Delay Time Optimization Control FIG. 10 is a flowchart illustrating control steps for delay time optimization in the flow cytometer 1. The control step includes a procedure of “calibration bead feeding step S ₁₁ ”, “signal acquisition step S ₁₂ ”, “optimum elapsed time search step S ₁₃ ”, and “delay time setting step S ₁₄ ”. Hereinafter, each procedure will be described.

(1) Calibration beads liquid feed step _{S 11}
When the analysis start signal is input by the user, the control unit 10 drives the pump of the tank unit that stores the sample liquid and the sheath liquid, and the sample liquid to the sample inlet 23 and the sheath inlet 24 of the microchip 2 and Start feeding the sheath liquid. The sample liquid includes the calibration beads described above. Further, the control unit 10 starts applying vibration to the orifice 21 by the vibration element 3. As a result, the three-dimensional laminar flow of the sample liquid and the sheath liquid ejected from the orifice 21 is discharged as droplets, and a fluid stream S is generated.

(2) Signal acquisition step S ₁₂ (second signal acquisition step)
In this step S _12, the droplets discharged from the orifice 21, and irradiated from the light source 71 a laser L _2, the detection of fluorescence generated from the droplets is performed. The laser L ₂ is irradiated to the droplet from the light source 71 after a predetermined time (Δt) from the time (T 0) when the calibration bead is detected by the irradiation detection unit, and the fluorescence generated from the droplet is detected by the detector 72. Is done.

Control unit 10 causes the emitted from the light source 71 a laser L ₂ from the time T0 after a different time. Specifically, the control unit 10, the time _{(T0 + Δt 1), (} T0 + Δt 2), is emitted from the light source 71 a laser _{L 2} in ··· (T0 + Δt _N). Here, N represents an integer of 3 or more. At each time, the detection intensity of the fluorescence detected by the detector 72 is output to the control unit 10 and held.

The detection intensity at the time (T0 + Δt _N ) is the detection intensity obtained by irradiating the droplet discharged from the orifice 21 with the laser L ₂ after the time Δt _{N has} elapsed since the time T 0 when the calibration bead was detected by the irradiation detection unit. Is an integrated value for M beads, for example.

(3) Optimal elapsed time search step S ₁₃
In this step S _13, the control unit 10, the integrated value of the detected intensity of the fluorescence generated from the droplets is greater (preferably the maximum value) to determine the elapsed time Delta] t _S. Specifically, the integrated values of the detected intensities acquired at times (T0 + Δt ₁ ) to (T0 + Δt _N ) are compared, and the elapsed time Δt _{S at} which the integrated value becomes larger is specified.

The elapsed time Δt _S is determined based on the sum of detection intensities of fluorescence generated from the beads A and B contained in the droplet. More preferably, the elapsed time Δt _S is determined based on the detection intensity of the fluorescence generated from the beads B (see the histogram of FIG. 7). The fluorescence generated from the bead A and the fluorescence generated from the bead B may be distinguished from each other according to the difference in the fluorescence wavelength, and only the fluorescence that matches the fluorescence wavelength of the bead B may be detected. Alternatively, the fluorescence generated from the beads B and the fluorescence generated from the beads B are detected by gating the beads according to the difference in size and detecting only the fluorescence generated from the beads within the selected size range (gate). Can also be distinguished.

(4) the delay time setting step _{S 14}
In this step S _14, calculated from the elapsed time Delta] t _s specified by the optimum elapsed time search step S ₁₃ the delay time ([Delta] T) is performed. Age Delta] t _s can be calibrated beads detected by the irradiation detector at time T0 is regarded as the time to pass through the irradiation position of the laser L ₂ emitted from the light source 71. Therefore, based on the elapsed time Delta] t _s, calibration beads detected by irradiation detection unit can calculate the delay time ([Delta] T) is the time to reach the break-off point. The control unit 10 sets the calculated delay time (ΔT) in the charging unit 41 so that charges are applied to the droplets at the optimum delay time.

  As described above, the flow cytometer 1 detects light generated from all types of calibration beads (here, beads A and B), or light generated only from types of beads having high fluorescence intensity (beads B). The delay time is optimized based on the strength. Thereby, optimization control of delay time can be performed using a strong detection signal. A stronger detection signal by keeping the population of beads (bead B) with larger size and higher fluorescence intensity in the calibration beads larger than the population of beads (bead A) with smaller size and lower fluorescence intensity This makes it possible to optimize the delay time using the.

  The flow cytometer 1 can perform the above-described optical position optimization control and delay time optimization control without replacing calibration beads. Conventionally, the position adjustment of the microchip with respect to the optical axis of the laser has been performed using beads with a small size, and it has been necessary to optimize the delay time separately using beads with high fluorescence intensity. On the other hand, in the flow cytometer 1, the calibration beads are exchanged for the optical position adjustment of the microchip and the optimization of the delay time by using calibration beads including two or more kinds of beads having different sizes and fluorescence intensities. It is possible to carry out easily without any problem.

The calibration method in the fine particle sorting apparatus according to the present technology can be configured as follows.
(1) A liquid feeding procedure for flowing a fluid containing two or more kinds of calibration particles having different sizes and fluorescence intensities through a flow path formed in the microchip or flow cell, and generated from the microchip or the flow cell by laser irradiation. The area average value of the first signal acquisition procedure for detecting the light to be detected from a plurality of positions on the microchip or the flow cell and the integrated value of the detection intensity of the light generated from all types of the calibration particles becomes larger. And a calibration method in a microparticle sorting apparatus, including a procedure for specifying a position.
(2) The calibration method according to (1), wherein an area average value of integrated values of detection intensities of light generated from all types of the calibration particles is used as the detection intensity.
(3) In the area where the area average value is larger, the method includes the step of identifying a first optimum position where the detection intensity of light generated from a small-sized seed particle among the calibration particles is larger. (2) The calibration method according to the description.
(4) The calibration according to (3), wherein the first optimum position is a position at which an integrated value or an average value of detected intensities of light generated from small-sized particles of the calibration particles is larger. Method.
(5) In an area where the area average value is larger, a second integrated value or a variation coefficient of the average value of the detected intensity of light generated from a small-sized particle of the calibration particles is smaller. The calibration method according to any one of (2) to (4), including a procedure for specifying an optimum position.
(6) The calibration method according to (5), including a procedure for setting a relative position of the microchip to the laser at the first optimum position or the second optimum position.
(7) The calibration method according to (6), wherein the relative position is set to the second optimum position when the first optimum position is different from the second optimum position.
(8) A laser beam is emitted to a droplet discharged from an orifice formed at one end of the flow path after a predetermined time has elapsed from the detection time of light generated from the calibration particles flowing through the flow path. A second signal acquisition procedure for detecting light generated from the droplet;
Identifies the elapsed time when the integrated value of the detection intensity of light generated from all types of the calibration particles or the integrated value of the detection intensity of light generated from particles of a large size among the calibration particles becomes larger A calibration method according to any one of the above (1) to (7).
(9) The calibration method according to (8), including a procedure for setting the elapsed time as a delay time.
(10) The calibration according to any one of the above (1) to (9), wherein two types of particles are used, which are particles having a small size and a low fluorescence intensity, and particles having a large size and a high fluorescence intensity. Method.
(11) The calibration method according to (10), wherein a population of particles having a large size and a high fluorescence intensity in the calibration particles is larger than a population of particles having a small size and a low fluorescence intensity.

In addition, the fine particle sorting apparatus according to the present technology may have the following configuration.
(1) An irradiation detection unit that detects light generated by irradiating a laser to a microchip or a flow cell in which fluids containing two or more kinds of calibration particles having different sizes and fluorescence intensities are passed through the flow path, and the irradiation A position adjustment unit that changes the relative position of the microchip or the flow cell with respect to the detection unit, and a movement signal to a position where the detection intensity of light generated from all types of calibration particles becomes larger is output to the position adjustment unit. A fine particle sorting device comprising a control unit.
(2) The fine particle sorting apparatus according to (1), wherein the detection intensity is an area average value of integrated values of detection intensity of light generated from all types of the calibration particles.
(3) In the area where the area average value is larger, the control unit is directed to the first optimum position where the detection intensity of light generated from the small-sized seed particle among the calibration particles is larger. The fine particle sorting apparatus according to (2), wherein a movement signal is output to the position adjustment unit.
(4) In the area where the area average value is larger, the control unit has an integrated value or a coefficient of variation of the average value of the detected intensity of light generated from a small-sized particle of the calibration particles. The fine particle sorting device according to (2) or (3) , wherein a movement signal to a second optimum position that decreases is output to the position adjustment unit.
(5) The control unit according to (4), wherein the control unit outputs a movement signal to the second optimum position to the position adjustment unit when the first optimum position and the second optimum position are different. Fine particle sorting device.
(6) a light source that irradiates a droplet discharged from an orifice formed at one end of the flow path with a laser after a predetermined time has elapsed from a detection time of light generated from the calibration particles by the irradiation detection unit; A detection unit that detects light generated from the droplets, and the control unit has an integrated value of detection intensities of light generated from all types of the calibration particles or a size of the calibration particles. The fine particle sorting device according to any one of (1) to (5), wherein an elapsed time when the integrated value of the detection intensity of light generated from a large type of particle becomes larger is set as a delay time.

Furthermore, the calibration particles according to the present technology may have the following configuration.
(1) Calibration particles for a microparticle sorting apparatus including two types of particles, which are small in size and low in fluorescence intensity, and large in size and high in fluorescence intensity.
(2) The calibration particle according to (1), wherein a population of the particles having a large size and a high fluorescence intensity is larger than a population of the particles having a small size and a low fluorescence intensity.
(3) The particle size of particles having a small size and low fluorescence intensity is 2 to 4 μm, and the particle size of particles having a large size and high fluorescence intensity is 8 to 12 μm. Calibration particles.

1: Microchip type microparticle sorting device, 2: Microchip, 21: Orifice, 22: Sample flow path, 23: Sample inlet, 3: Vibrating element, 41: Charging part, 42: Electrode, 51, 52: Deflection Plate, 61, 71: Light source, 62, 72: Detector, 81, 82, 83: Collection container, 9: Position adjustment unit, 10: Control unit, D: Detection position, F ₁ , F ₂ : Fluorescence, L ₁ , L ₂ : Laser

Claims (14)

A liquid feeding procedure for flowing a fluid containing two or more kinds of calibration particles having different sizes and fluorescence intensities through a flow path formed in a microchip or a flow cell;
A first signal acquisition procedure for detecting light generated from the microchip or the flow cell by laser irradiation from a plurality of positions on the microchip or the flow cell;
A step of detecting the intensity of light to identify the larger a position generated from all species of the calibration particles, only including,
As the detection intensity, using the area average value of the integrated value of the detection intensity of light generated from all types of the calibration particles,
In the area where the area average value is larger, the fine particle sorting including a procedure for identifying the first optimum position where the detection intensity of light generated from the small-sized seed particle among the calibration particles becomes larger Calibration method in the apparatus. It said first optimum position calibration method of claim 1, wherein the integrated value or the average value of the detected intensity of light is greater a position generated from a small seed particles size of the calibration particle. In the area where the area average value is larger, a second optimum position where the integrated value of the detection intensity of light generated from the particles of the small size among the calibration particles or the coefficient of variation of the average value becomes smaller calibration method according to claim 1 or 2, wherein including specific procedures. The calibration method according to claim 3 , further comprising a step of setting a relative position of the microchip or the flow cell with respect to the laser to the first optimum position or the second optimum position. The calibration method according to claim 4 , wherein the relative position is set to the second optimum position when the first optimum position and the second optimum position are different. A liquid is emitted to a droplet discharged from an orifice formed at one end of the flow path after a predetermined time has elapsed from a detection time of light generated from the calibration particles flowing through the flow path, and the liquid A second signal acquisition procedure for detecting light generated from the drop;
Identifies the elapsed time when the integrated value of the detection intensity of light generated from all types of the calibration particles or the integrated value of the detection intensity of light generated from particles of a large size among the calibration particles becomes larger The calibration method according to any one of claims 1 to 5 , further comprising: The calibration method according to claim 6 , further comprising a step of setting the elapsed time as a delay time. The calibration method according to any one of claims 1 to 7 , wherein two types of particles are used as the calibration particles: particles having a small size and low fluorescence intensity and particles having a large size and high fluorescence intensity. The calibration method according to claim 8 , wherein a population of particles having a large size and a high fluorescence intensity in the calibration particles is larger than a population of particles having a small size and a low fluorescence intensity. 10. The calibration method according to claim 8, wherein the particle size of the particles having a small size and low fluorescence intensity is 2 to 4 μm, and the particle size of the particles having a large size and high fluorescence intensity is 8 to 12 μm. An irradiation detection unit that detects light generated by irradiating a laser to a microchip or a flow cell in which fluids containing two or more kinds of calibration particles having different sizes and fluorescence intensities are passed through the flow path;
A position adjusting unit for changing the relative position of the microchip or the flow cell with respect to the irradiation detecting unit;
A control unit that outputs to the position adjustment unit a movement signal to a position where the detection intensity of light generated from all types of the calibration particles becomes larger , and
The detection intensity is an area average value of integrated values of detection intensity of light generated from all types of the calibration particles,
In the area where the area average value is larger, the control unit outputs a movement signal to a first optimum position where the detection intensity of light generated from a small-sized particle among the calibration particles becomes larger. A fine particle sorting device for outputting to the position adjusting unit . In the area where the area average value is larger, the control unit has a smaller integrated value or a variation coefficient of the average value of the detection intensity of light generated from a kind of particle having a smaller size among the calibration particles. The fine particle sorting device according to claim 11 , wherein a movement signal to a second optimum position is output to the position adjusting unit. The fine particle sorting according to claim 12 , wherein the control unit outputs a movement signal to the second optimum position to the position adjustment unit when the first optimum position and the second optimum position are different. apparatus. A light source that irradiates a laser after a predetermined time has elapsed from a detection time of light generated from the calibration particles by the irradiation detection unit with respect to a droplet discharged from an orifice formed at one end of the flow path,
A detection unit for detecting light generated from the droplets,
The control unit has a larger integrated value of detection intensity of light generated from all types of calibration particles, or an integrated value of detection intensity of light generated from particles of a large size among the calibration particles. The minute particle sorting device according to any one of claims 11 to 13 , wherein the elapsed time is set as a delay time.

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